1. Field
This disclosure is concerned generally with a biological assay useful for determining the potency of antigenic preparations and specifically with determining the antigenicity or potency of preparations useful for immunizing animals against bacteria of the genus Streptococcus, especially group C streptococcal organisms such as Streptococcus equi.
2. Prior Art
Organisms of the genus Streptococcus include a variety of nonmotile, chiefly parasitic, gram-positive bacteria that divide only in one plane, occur in pairs or chains, but not packets, and include important pathogens of man and domestic animals. The streptococcal genus has been divided into eight distinct groups labeled A, B, C, D, F, G, H, and K. It is known that Group A and B human Streptococci have an affinity for heart muscle causing rheumatic fever, endocarditis, and sometimes death. A Group C organism, Streptococcus equi, is the causitive agent of a severe respiratory disease of horses referred to as "Strangles". The disease is endemic in most parts of the world and epidemic in the United States. Race and show horses are particularly susceptible to repeated infections due to the stress of travel and exposure to new contacts. The disease begins with a mucopurulent nasal discharge, temperatures of 103.degree.-106.degree. F., and severe inflammation of the upper respiratory mucosa. It finally progresses to lymphadenitis and abscess formation which is sometimes severe enough to restrict air intake and cause suffocation of the animal. Strangles results in extensive loss of condition (loss of weight) as it often runs a course of 4-6 weeks. The single strain of Streptococcus equi (a Lancefield Type C Strep.) is responsible for this disease world-wide. See for example, Bergy's Manual of Determinitive Bacteriology (8th Edition), p. 498 (1974). The only other known susceptible animal is the mouse.
Because of the debilitating and in some cases lethal effects of streptococcal infections in man and other animals, attempts have been made over the years to prepare streptococcal bacterins or bacterin-like preparations which could be used for active immunization purposes. Unfortunately, some streptococcal preparations tend to be very reactive and it has been noted that human streptococcal "vaccines" have stimulated heart muscle reactions while Strep. equi preparations, in live or inactivated forms, have been noted for their affinity for dermal tissue, producing severe swelling at the injection site. These known reactivities have tended to discourage the development and/or commercial use of immunizing streptococcal products for man and other animals. Strep. equi ("M-protein extraction") preparations are available and have been described in U.S. Pat. No. 3,793,150 and U.S. Pat. No. 3,852,420. The purification of such M-like proteins is also described in an article by J. B. Woolcock, Infection & Immunity, July, 1974, p. 116-122. Although there does exist a whole culture, chemically inactivated, Strep. equi preparation (Ft. Dodge Corp.), the product appears to provide only minimal reduction in Strangles disease symptoms and produces significant swellings and stiffness due to the high reactivity of that bacterin. This has resulted in relatively modest acceptance of the product.
In efforts to prepare a non-reactive and efficacious streptococcal vaccine in general and a Streptococcus equi vaccine in particular, it would be highly desirable to have available an accurate assay for determining the potency of the final streptococcal preparation since a commercial streptococcal preparation should be, ideally, not only non-reactive, but it should also have a reliable and quantifiable potency or antigenicity. In our efforts to prepare a Streptococcus equi vaccine, we soon became aware that there was no laboratory method (as opposed to in vivo testing in horses per se) for determining antigenicity. Thus, it became quite important to devise a reliable method, other than simple horse challenge, to quantitate immunogenicity of a given preparation. Quite surprisingly, we found that such a method is possible, thus obviating the use of horses for routine antigen potency testing. Although our indirect method of antigen quantitation has been found especially useful in evaluating a specific Group C streptococcal antigen preparation derived from Strep. equi, it is thought that our method is applicable for determining the potency of all Streptococcal antigen preparations. Details of our potency determination method are disclosed below.